一种新型淋球菌表面表达抗原介导的保护性免疫应答极化的研究

Neisseria gonorrhoeae has developed resistance against most of the commonly used antibiotics, which poses a major treat for future managing and treatment of gonorrhea infections. However, developing a vaccine against N. gonorrhoeae is in particular complicated by fact that there are no known correlates of protection and a natural infection does not result in a significant adaptive immune response. Also, surface expressed proteins show extremely high sequence variability. Although little is known about the mechanisms N. gonorrhoeae exploits to suppress an adaptive immune response, recent breakthroughs indicated that specific induction of the Th1 response during primary infection of a mouse model with N. gonorrhoeae resulted in a memory response, clearance of the infection and protection against reinfection. These results indicate that Th1-inducing adjuvants might be essential for eliciting protective memory responses against vaccine antigens. But, whether they can really enhance Th1 response induced by antigen and also have the protection effect needs to be verified. In previous research of the applicant on vaccine development against Neisseria meningitidis, a molecular adjuvant was used that was able to skew an immune response in the direction of a Th1 response. This molecular adjuvant (IMX315) is the oligomerisation domain of C4b binding protein. Fusion proteins between this molecular adjuvant and the target protein antigen (fHbp) showed induced immunogenicity in a mouse model and particularly an enhanced Th1 response. Furthermore, serum raised by these mice showed enhanced bactericidal activity against N. meningitidis. To achieve these goals, conservation of all putative surface expressed proteins will be compared using the 16 available fully sequenced N. gonorrhoeae genomes. Surface expression and accessibility of the best candidate such as MtrE will be verified and the purified protein or peptide will be formulated with various Th1 stimulating adjuvant such as Toll-like receptor agonists. Vaccine formulations will be investigated for stimulating Th1 responses in in vitro cellular assays. The best candidate Th1 stimulating formulations will be used for immunogenicity studies in mice to investigate whether Th1 polarization is also observed in vivo. Mice will be analysed for both cellular and (functional) antibody responses. The best vaccine formulations will subsequently be used in mouse vaginal infection models to determine whether the Th1 responses elicited by the vaccines are functional and able to clear an infection when administered therapeutically. In addition, the vaccine will also be investigated for their ability to provide prophylactic protection against an infection. Using this strategy, this project will establish whether the Th1 response is correlate of protection and whether future N. gonorrhoeae infections could be treated with therapeutic Th1 stimulating vaccines.

淋球菌对大部分抗生素具有耐药性，表面蛋白具高度变异性且抑制获得性免疫的机制知之甚少，疫苗研发十分困难。最近研究指出淋球菌感染小鼠模型中诱导Th1型免疫应答，可清除和预防感染，提示Th1诱导佐剂对疫苗保护性的必要性，但是否增强激发淋球菌抗原的Th1应答极化并产生保护作用有待证实。我们前期研究发现佐剂IMX315蛋白显著激发脑膜炎球菌抗原的Th1应答并提高杀菌活性，另外已比较分析16个已知全序列淋球菌基因组中表面蛋白保守性，筛选出可及性最好的新型淋球菌表面抗原蛋白（如MtrE）。本课题在此基础上，将IMX315、TLR激动剂与蛋白进行融合或混合。体外和体内实验研究其Th1型应答刺激、免疫原性和功能性抗体反应。利用小鼠阴道感染模型检测佐剂-抗原蛋白导致的Th1型应答能否清除感染和对再次感染的保护。将揭示Th1诱导佐剂-抗原蛋白能否诱导Th1型极化并产生保护，为未来开发新型Th1刺激性疫苗奠定基础

多药耐药性淋病奈瑟菌（淋球菌）菌株的增加对卫生保健构成了威胁，然而由于淋球菌表面表达蛋白的抗原可变性、阶段变异性以及淋球菌感染引发的免疫保护机制尚不明确，有效疫苗的开发仍然难以实现。然而，近期研究表明，Th1极化免疫反应可能对构建淋球菌疫苗有益。因此，本项目的目标是鉴定一种合适的具有广泛保护性的保守表面表达抗原，为该表面抗原寻找一种合适的Th1极化佐剂，并确定这种新抗原/佐剂制剂作为淋球菌疫苗的预防性和治疗性功效。本项目利用In silico方法预测了淋球菌所有表面表达蛋白，并分析了表面蛋白的序列可变性和阶段变异性。MtrCDE多药耐药外排泵的外膜通道蛋白MtrE及其表面表达的Loop2肽段被鉴定为最为保守的表面表达蛋白。体内实验表明，在小鼠阴道感染模型中，野生型细菌竞争能力远远强于MtrCDE缺失突变株。Loop2肽段和IMX315分子佐剂（C4b结合蛋白寡聚区域）组成的两种不同的融合蛋白已成功构建并纯化，即Nlloop2(Loop2为线性表位)及Intraloop2(Loop2为结构表位)。MtrE、Intraloop2、Nlloop2均可引起较高的蛋白特异性抗体滴度，值得一提的是，Intraloop2和Nloop2具有极高的抗Loop2滴度。此外，针对上述三种蛋白的抗体都能够识别淋球菌表面表达的MtrE，并显示出高度的MtrE依赖性杀菌活性。随后的体外树突状细胞激活实验和T细胞极化实验证实，与上述抗原联合使用后，CpG 1826是最有效的Th1极化佐剂。CpG 1826疫苗制剂再次引起较高的蛋白特异性抗体滴度，Intraloop2和Nloop2显示极高的抗Loop2滴度。此外，抗体具有杀菌作用并能够干扰MtrCDE外排泵活性。同时，在小鼠阴道感染模型中，MtrE、Intraloop2和Nloop2疫苗均能对淋球菌感染提供保护，Intraloop2和Nloop2的疫苗效果优于MtrE。此外，MtrE、Intraloop2或Nloop2作为治疗性疫苗也能加速小鼠阴道感染模型中淋球菌的清除，Intraloop2和Nloop2的治疗性疫苗效果也优于MtrE。综上所述，本研究证实MtrE及其Loop2是完全保守的新型表面抗原，上述抗原与Th1极化佐剂CpG 1826联合使用，具有较好的的预防性和治疗性功效。